The short-wavelength reactive dyes that we recommend for preparing the brightest blue-fluorescent bioconjugates are the Alexa Fluor 350, Alexa Fluor 405, AMCA-X, Marina Blue, Pacific Blue and Cascade Blue derivatives (Amine-reactive, ultraviolet light-excitable fluorophores for labeling proteins and nucleic acids—Table 1.12). Alexa Fluor 430, Pacific Orange and Cascade Yellow dyes fill a spectral void because they exhibit the rare combination of absorption between 400 nm and 450 nm and fluorescence emission beyond 500 nm. The amine-reactive naphthalene, pyrene and Dapoxyl derivatives are important for the production of environment-sensitive probes in protein structure and function studies (Amine-reactive, environment-sensitive fluorophores—Table 1.13); their thiol-reactive counterparts are discussed in Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3. Many of our UV light–excitable reactive dyes are more commonly employed for such bioanalytical techniques as HPLC derivatization, amino acid sequencing and protein determination and are therefore discussed in Reagents for Analysis of Low Molecular Weight Amines—Section 1.8.

Derivatives of 7-aminocoumarin dyes are widely used labeling reagents for preparing protein and nucleic acid conjugates, and we offer the amine-reactive 7-aminocoumarin derivatives Alexa Fluor 350 carboxylic acid succinimidyl ester (A10168).

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The Zenon Alexa Fluor 350, Zenon Alexa Fluor 430 and Zenon Pacific Blue Antibody Labeling Kits (Zenon Antibody Labeling Kits—Table 7.7) permit the rapid and quantitative labeling of antibodies—even submicrogram amounts—using a purified antibody fraction or a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. These kits, along with Zenon technology, are described in detail in Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3.

Conjugates of two isomers of dansyl chloride (2,5-dansyl chloride and 2,6-dansyl chloride) have smaller Stokes shifts and appreciably longer fluorescence lifetimes (up to ~30 nanoseconds) than conjugates of 1,5-dansyl chloride, making these isomers among the best available probes for fluorescence depolarization studies. These dyes are particularly useful for preparing fluorescent drug or ligand analogs that are expected to bind to hydrophobic sites in proteins or membranes. The lipophilicity of these reagents may also facilitate the labeling of sites within the membrane-spanning portions of cellular proteins.

As with Cascade Blue acetyl azide, Alexa Fluor 405 dye shows minimal spectral overlap with green fluorophores, making it ideal for multicolor applications. However, the violet fluorescence of Cascade Blue and Alexa Fluor 405 dyes is less visible to the human eye in fluorescence microscopy applications than the blue fluorescence of Alexa Fluor 350 and AMCA-X dyes. Alexa Fluor 405 dye is available as:

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Figure 1.7.4 Normalized fluorescence emission spectra of Cascade Blue (CB), 7-amino-4-methylcoumarin (AMC) and fluorescein in aqueous solutions.

As compared with the aminocoumarin derivatives, the Cascade Blue fluorophore shows less spectral overlap with fluorescein (Figure 1.7.4), an important advantage for multicolor applications. In addition, this reactive Cascade Blue derivative has high absorptivity, is highly fluorescent and resists quenching upon protein conjugation (Figure 1.7.5). Even at low degrees of labeling, Cascade Blue conjugates are significantly more fluorescent than are those of 7-amino-4-methylcoumarin-3-acetic acid (AMCA), and they remain preferred reagents for multicolor flow cytometry.

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Marina Blue and Pacific Blue dyes, both of which are based on the 6,8-difluoro-7-hydroxycoumarin fluorophore, exhibit bright blue fluorescence emission near 460 nm (Amine-reactive, ultraviolet light-excitable fluorophores for labeling proteins and nucleic acids—Table 1.12). The Marina Blue dye is optimally detected using optical filters configured for DAPI, whereas the Pacific Blue dye is ideally suited for 405 nm violet diode laser excitation on the Applied Biosystems Attune acoustic focusing cytometer and similarly equipped fluorescence microscopes (Figure 1.7.7). Significantly, the pKa values of these 6,8-difluoro-7-hydroxycoumarin derivatives are 2–3 log units lower than those of the corresponding 7-hydroxycoumarins (Figure 1.7.3). Thus, the Marina Blue and Pacific Blue dyes yield conjugates that are strongly fluorescent, even at neutral pH. For preparing bioconjugates, we offer the amine-reactive succinimidyl ester of the Pacific Blue dye (P10163).

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Dansyl chloride is nonfluorescent until it reacts with amines. The resulting dansyl amides have environment-sensitive fluorescence quantum yields and emission maxima, along with large Stokes shifts. Despite the weak absorptivity (ε ~4000 cm-1M-1 at 330–340 nm) and moderate fluorescence quantum yield of dansyl sulfonamides, dansyl chloride is widely used as a derivatization reagent for end-group analysis of proteins, amino acid analysis and HPLC detection (Reagents for Analysis of Low Molecular Weight Amines—Section 1.8). The succinimidyl ester of dansylaminohexanoic acid (dansyl-X, SE) contains a seven-atom spacer ("X") that places the dansyl fluorophore further from its reaction site, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and enhancing accessibility to antibody binding. A rabbit polyclonal antibody to the 1,5-dansyl fluorophore (A6398) that significantly enhances the dye's fluorescence is described in Anti-Dye and Anti-Hapten Antibodies—Section 7.4.

We also prepare Alexa Fluor 405 conjugates of secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Alexa Fluor 405 conjugates are recognized by the anti–Alexa Fluor 405/Cascade Blue dye antibody (A5760, Anti-Dye and Anti-Hapten Antibodies—Section 7.4).

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Conjugates of the amino-reactive succinimidyl ester of 1-pyrenebutanoic acid (P130)  have exceptionally long excited-state lifetimes (sometimes >100 nanoseconds), relatively short-wavelength emission and capacity for proximity-dependent excimer formation (Figure 1.7.6). These amine-reactive pyrene derivatives have primarily been used for labeling and detecting oligonucleotides, biogenic amines and polyamines. Pyrene binds strongly to carbon nanotubes via pi-stacking interactions. This property makes 1-pyrenebutanoic acid succinimidyl ester (P130) a valuable reagent for functionalizing these remarkable nanomaterials for coupling to proteins.

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The long fluorescence lifetime of pyrenebutyric acid (1-pyrenebutanoic acid) permits time-gating of the fluorescence, which is a useful technique for discriminating between the dye signal and sample autofluorescence, and has been exploited for fluorescence immunoassays. For preparing pyrene conjugates with long fluorescence lifetimes, we recommend the more water-soluble succinimidyl ester of N-(1-pyrenebutanoyl)cysteic acid. The amine-reactive 1-pyrenesulfonyl chloride has been used to generate a fluorescent ATP sensor via modification of an ATP-binding ribonucleopeptide.

Bimane mercaptoacetic acid (carboxymethylthiobimane) is a blue-fluorescent dye with excitation/emission maxima of ~380/458 nm. It is useful as a reference standard for the fluorogenic monobromobimane and monochlorobimane reagents (Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) because it is an analog of the thioether product of their reaction with glutathione and other thiols.

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Figure 1.7.1 Comparison of the relative fluorescence of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) streptavidin () and Alexa Fluor 350 streptavidin, a sulfonated AMCA derivative (S11249, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein.

Like the Alexa Fluor 430 and Pacific Blue dyes described above, the Cascade Yellow dye exhibits an excitation maximum that falls between those of the UV light–excited dyes and the fluoresceins. This sulfonated pyridyloxazole (PyMPO) laser dye exhibits an absorption maximum near 410 nm and an unusually high Stokes shift, with relatively strong emission at 550–570 nm. The large Stokes shift permits detection at a wavelength well beyond that of most sample autofluorescence, and allows multiple fluorophores to be excited at the same wavelengths and detected at different wavelengths. For example, protein conjugates of Cascade Yellow succinimidyl ester can be simultaneously excited at 405 nm with Pacific Blue conjugates, and then separately detected at longer wavelengths (Figure 1.7.7). Cascade Yellow and Cascade Blue antibody conjugates, along with several phycobiliprotein tandem conjugates, are utilized in an 11-color polychromatic flow cytometry technique.

Figure 1.7.5 Histograms showing the fluorescence per fluorophore for A) fluorescein and B) Cascade Blue conjugated to various proteins, relative to the fluorescence of the free dye in aqueous solution, represented by 100 on the y-axis. The proteins represented are: 1) avidin, 2) bovine serum albumin, 3) concanavalin A, 4) goat IgG, 5) ovalbumin, 6) protein A, 7) streptavidin and 8) wheat germ agglutinin.

Few reactive dyes that absorb between 400 nm and 450 nm have appreciable fluorescence beyond 500 nm in aqueous solution. Alexa Fluor 430 dye fills this spectral gap. Excitation near its absorption maximum at 431 nm (Figure 1.7.2) is accompanied by strong green fluorescence with an emission maximum at 541 nm. The amine-reactive succinimidyl ester of Alexa Fluor 430 carboxylic acid (A10169) is available, as well as Alexa Fluor 430 conjugates of secondary antibodies (A11063, A11064; Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (S11237, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9).

Cascade Blue acetyl azide is the amine-reactive sulfonated pyrene derivative that we use to prepare  blue-fluorescent Cascade Blue dye–labeled proteins and dextrans. The polar nature of this reagent makes it difficult to purify to homogeneity; however, we offer a Cascade Blue acetyl azide preparation (C2284, Amine-reactive, ultraviolet light-excitable fluorophores for labeling proteins and nucleic acids—Table 1.12) that is ~60% reactive and packaged according to the net weight of the reactive dye. The remaining constituents are inorganic salts or unreactive forms of the dye that can readily be removed following conjugation.

Shorter-wavelength amine-reactive fluorophores are less frequently used for preparing bioconjugates because dyes excited with longer wavelengths, and therefore lower energy, are widely available and less likely to cause photodamage to labeled biomolecules. Moreover, many cells and tissues autofluoresce when excited with ultraviolet (UV) light, producing detection-confounding background signals. However, for certain multicolor fluorescence applications—including immunofluorescence, nucleic acid and protein microarrays, in situ hybridization and neuronal tracing—a blue-fluorescent probe provides a contrasting color that is clearly resolved from the green, yellow, orange or red fluorescence of the longer-wavelength probes.

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Aminonaphthalene-based probes tend to have emission spectra that are sensitive to the environment and to exhibit weak fluorescence in aqueous solution. Spectra of environment-sensitive probes respond to perturbations in the local environment (Amine-reactive, environment-sensitive fluorophores—Table 1.13). For example, changes in solvation that occur because of ligand binding, protein assembly or protein denaturation can often evoke changes in the fluorescence properties of these probes. This property has made dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) and other aminonaphthalene-based dyes important tools for protein structural studies.

We have also exploited the environment-sensitive fluorescence of the Dapoxyl dye (Figure 1.7.8) to develop a highly selective and photostable stain for the endoplasmic reticulum (ER-Tracker Blue-White DPX, E12353; Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4).

The hydroxycoumarins exhibit pH-sensitive spectral properties, but the methoxycoumarins do not. Hydroxycoumarins are often used to prepare reactive intermediates for the synthesis of radioiodinated materials. The spectral properties of the hydroxycoumarins allow their quantitation prior to radioiodination.

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For easy and trouble-free labeling of proteins with succinimidyl esters of the Alexa Fluor 350, Alexa Fluor 430 and Pacific Blue dyes, we offer Alexa Fluor 350 and Pacific Blue Protein Labeling Kits (A10170, P30012; Active esters and kits for labeling proteins and nucleic acids—Table 1.2). These kits, which are described in greater detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2, contain everything that is required to perform three separate labeling reactions and to purify the resulting conjugates (Molecular Probes kits for protein and nucleic acid labeling—Table 1.3). The Alexa Fluor 350 and Pacific Bue Antibody Labeling Kits (A20180, P30013) can be used to prepare blue-fluorescent conjugates of monoclonal and polyclonal antibodies, as well as of other proteins in limited quantities (five labeling reactions of ~100 µg each). The APEX Pacific Blue Antibody Labeling Kit (A10478) utilizes a solid-phase technique to label 10–20 µg IgG antibody, even in the presence of stabilizing proteins or amine-containing buffers.

The pyridyloxazole derivatives—including the succinimidyl ester (PyMPO, SE; S6110)—fill the spectral gap between UV light–excited dyes and the fluoresceins. These derivatives of the laser dye PyMPO exhibit absorption maxima near 415 nm and unusually high Stokes shifts, with emission at 560–580 nm. Like the naphthalene-based dyes, the pyridyloxazole dyes exhibit environment-sensitive fluorescence spectra. PyMPO SE has been used to synthesize fluorescent gramicidin derivatives for following ion channel–gating processes.

Figure 1.7.6 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.

With excitation/emission maxima of 402/421 nm (Figure 1.7.2), Alexa Fluor 405 dye is well matched to the 405 nm spectral line of violet diode lasers for fluorescence microscopy and flow cytometry. Alexa Fluor 405 succinimidyl ester is an amine-reactive derivative of our Cascade Blue dye. Not only is it offered at higher purity than the alternative Cascade Blue acetyl azide, but Alexa Fluor 405 succinimidyl ester also contains a 4-piperidinecarboxylic acid spacer that separates the fluorophore from its reactive moiety. This spacer enhances the reactivity of the succinimidyl ester and minimizes any interactions between the fluorophore and the biomolecule to which it is conjugated.

The succinimidyl ester of the Pacific Orange dye (P30253) yields conjugates with excitation/emission maxima of ~400/551 nm, making it ideal for use with 405 nm violet diode laser–equipped flow cytometers and fluorescence microscopes. Moreover, Pacific Blue and Pacific Orange conjugates can be simultaneously excited at 405 nm and emit at 455 nm and 551 nm, respectively, facilitating two-color analysis.

Several of our kits facilitate protein labeling with the Pacific Orange succinimidyl ester, including the Pacific Orange Antibody Labeling Kit (P30014) and the Zenon Antibody Labeling Kits (Zenon Antibody Labeling Kits—Table 7.7), which are described in greater detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

The sulfonated coumarin derivative, Alexa Fluor 350 carboxylic acid succinimidyl ester, is more water soluble than either AMCA succinimidyl ester or AMCA-X succinimidyl ester and yields protein conjugates that are more fluorescent than those prepared from its nonsulfonated analog (Figure 1.7.1). Alexa Fluor 350 protein conjugates are optimally excited at 346 nm and have bright blue fluorescence emission (Figure 1.7.2, ) at wavelengths slightly shorter than AMCA or AMCA-X conjugates (442 nm versus 448 nm), which reduces the dye's spectral overlap with the emission of fluorescein. We offer several reactive versions of Alexa Fluor 350 dye, including:

Figure 1.7.3 Comparison of the pH-dependent fluorescence responses of 7-hydroxy-4-methylcoumarin and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566). Fluorescence intensities were measured for equal concentrations of the two dyes using excitation/emission at 355/460 nm. Compared with 7-hydroxy-4-methylcoumarin (pKa = 7.8), DiFMU (pKa = 4.9) provides superior detection sensitivity in the 5–8 pH range under these conditions.

Dapoxyl dye is a particularly versatile derivatization reagent and precursor to environment-sensitive probes. Like Cascade Yellow dye, Dapoxyl dye exhibits an exceptionally large Stokes shift, with excitation/emission maxima of ~370/580 nm. Sulfonamides from Dapoxyl sulfonyl chloride have much higher extinction coefficients than those of dansyl chloride (~26,000 cm-1M-1 versus about 4000 cm-1M-1) and equal or greater quantum yields when dissolved in organic solvents; however, the fluorescence of Dapoxyl derivatives is very sensitive to the dye environment, and fluorescence in water is very low, making them useful for sensing conformational changes, denaturation and phosphorylation states of proteins.